Functional Characterization of Rare Variants in the Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation.

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended gene analysis in patients with distinct arrhythmias, we investigated as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated and , together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving . Moreover, our data highlight the importance of functional investigations of rare variants.